DNA molecules having human bcl-2 gene sequences

ABSTRACT

The sequence of the protein coding regions of the bcl-2 gene are provided as well as bacterial clones which produce the proteins. Assays are provided for detecting a class of B-cell neoplasms associated with a chromosome translocation between chromosomes 14 and 18. This translocation is involved in the majority of cases of human follicular lymphomas. One assay employs an antibody which is immunoreactive with a human protein which is over-expressed due to the chromosome translocation. Another assay involves measurement of the amount of mRNA which hybridizes to the gene proximal to the translocation break-point.

This invention was made with government support under Grant CA 39860from the National Cancer Institute. The United States Government hascertain rights in this invention.

This application is a continuation of application Ser. No. 07/994,941,filed Dec. 23, 1992, which is a continuation of application Ser. No.663,010 filed Mar. 19, 1991, now U.S. Pat. No. 5,202,429, which is acontinuation of application Ser. No. 883,687, filed Jul. 9, 1986, nowU.S. Pat. No. 5,015,568.

BACKGROUND OF THE INVENTION

Specific chromosomal rearrangements, predominantly translocations andinversions, are observed in the great majority of human hematopoieticmalignancies. In Burkitt lymphoma the specific chromosomaltranslocations result In the juxtaposition of one of the three humanimmunoglobulin loci and the c-myc oncogene. In diffuse B-cell lymphomas,multiple myelomas and chronic lymphocytic leukemias of the B-cell typecarrying the t(11;14) (q13;q32) chromosome translocation, the bcl-1locus is translocated to the heavy-chain locus on chromosome 14. In mostcases of follicular lymphoma, one of the most common human hematopoieticmalignancies, a (t14;18) (q32; q21) chromosome translocation has beenobserved. This translocation moves the bcl-2 gene to a position adjacentto the heavy-chain locus. In one cell line derived from a leukemicpatient having both a t(14;8) and a t(14;18) translocation enhanced mRNAproduction from the bcl-2 gene was observed. (Tsujimoto et al, Science,Vol. 228, pages 1440-1443 (1885).) It was concluded there that thetranscription unit of the bcl-2 gene spans the chromosome break-point,and thus the oncogene protein is likely to be structurally altered inthe B-cell neoplasms. Surprisingly, it has now been found that thetranslocation does not alter the oncogene protein itself, as thetranslocation break-points occur downstream from the actual proteincoding sequences. Thus oncogenesis may be solely due to theoverproduction of the normal human gene products of the bcl-2 gene.

Effective treatment for cancer is often dependent upon an early andproper diagnosis of the malignancy. There is thus a need for simple andaccurate diagnostic methods for detecting and identifying humanmalignancies, such as follicular lymphomas, in general, and follicularlymphomas in particular.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a diagnostic methodfor detecting B-cell neoplasms having t(14;18) translocations in ahuman.

It is another object of the present invention to provide a human bcl-2gene which can be expressed in bacteria.

It is yet another object of the present invention to provide asubstantially pure protein preparation of a bcl-2 gene product.

It is still another object of the present invention to provide adiagnostic method of detecting B-cell neoplasms employing an antibodywhich is immunoreactive with a bcl-2 gene product.

These and other objects of the invention are achieved by one or more ofthe following embodiments.

In one embodiment a diagnostic method for detecting B-cell neoplasms ina human is provided, comprising the steps of:

isolating B-cells from the human;

extracting proteins from said B-cells to form a test sample;

contacting said test sample with an antibody which is immunoreactivewith a gene product of the bcl-2 gene, under conditions Whereantibody-antigen complexes are formed and are stable;

quantitating the amount of antibody-antigen complexes formed with saidtest sample; and

comparing the amount of antibody-antigen complexes formed with said testsample to the amount formed with a control sample of proteins, a ratioof test sample complexes to control sample complexes of greater thanabout ten indicating B-cell neoplasm, said control sample of proteinsextracted from cells selected from the group consisting of: B-cells froma normal human, cells from an established normal B-cell or pre- B-cellline, and non-B-cells from said human.

Also provided are forms of the human bcl-2 gene which are substantiallyfree of introns. Such genes can be replicated and expressed in bacteriato form proteins having the same primary structure as the bcl-2 proteinsproduced in humans.

Also provided by the present invention are substantially purepreparations of proteins having an N-terminal end encoded by the firstexon of the human bcl-2 gene.

In yet another embodiment of the present invention a diagnostic methodfor detecting B-cell neoplasms in a human is provided comprising thesteps of:

isolating B-cells from the human;

extracting RNA from said B-cells to form a test sample;

contacting said test sample with a DNA probe containing a sequence of atleast 15 neucleotides in length derived from the human bcl-2 gene, underconditions where homologous RNA-DNA hybrids form and are stable;

quantitating the amount of RNA-DNA hybrids formed with the test sample;and

comparing the amount of RNA-DNA hybrids formed with said test sample tothe amount formed with a control sample of RNA, a ratio of test samplehybrids to control sample hybrids of greater than about ten indicatingB-cell neoplasm, said control sample of RNA extracted from cellsselected from the group consisting of: B-cells from a normal human,cells from a normal B-cell or pre-B-cell line, and non-B-cells from saidhuman.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a genomic restriction map of chromosome 18, and thestructure of cDNA clones.

FIG. 2 shows the nucleotide sequence of bcl-2 cDNA corresponding to the5.5 kb transcript. Only the sequences surrounding the open reading frameare shown.

FIG. 3 shows the nucleotide sequence of bcl-2 cDNA corresponding to the3.5 kb transcript. Only the sequences surrounding the open reading frameare shown.

DETAILED DESCRIPTION OF THE INVENTION

According to one aspect of the present invention it has been discoveredthat B-cell neoplasms which are associated with t(14;18) chromosometranslocations cause an increase in the expression of both the mRNA andthe protein products of the bcl-2 gene. The expression in the neoplasticB-cells is generally about ten-fold higher than the amount expressed bynormal cells. This elevated expression can be used as a diagnostic toolto detect the B-cell neoplasms in humans. Such neoplasms includefollicular lymphomas as well as other lymphomas.

It has been found that three species of mRNA are transcribed from thebcl-2 gene. At least two different protein products have beenidentified. A 239 amino acid protein, designated bcl-2-alpha istranslated from a 5.5 kb mRNA. A protein of 205 amino acids, bcl-2-betais translated from a 3.5 kb mRNA. It is thought that bcl-2-alpha is alsotranslated from the 8.5 kb mRNA species. All three size species oftranscript share substantial sequence homology in the 5' portion of thegene, termed the first exon or 5' exon. The larger two transcriptsappear to be spliced to a second exon which is at least 50 kb distal tothe first. The splice site is in the midst of the protein codingsequence. Thus, the 3.5 kb transcript codes for a protein having adifferent carboxyl terminmus than the proteins coded by the two largersized transcripts.

The hotspot for chomosome break-points among the t(14;18) translocationsof follicular lymphomas map 3' to the protein coding region. Thereforeit has now been found that the translocations do not alter the primarystructure of the protein products.

Bacterial isolates available from the ATCC, under Accession Numbers67147 and 67148, can be used to express bcl-2 gene products alpha andbeta, respectively, in bacteria. The clones of the bcl-2 gene wereobtained via cDNA cloning and so do not contain introns. Thus theseclones can be expressed in bacteria to make products having the sameprimary sequence as those made in the human body. After growing thebacteria under suitable-conditions which are well known in the art, thecells can be harvested and disrupted to extract total cellular protein.The protein can then, for example, be placed on a sizing column such asSepharos™ or agarose beads, and proteins of the correct molecularweight, i.e., between about 20 and 30 kD can be collected.

Further purification can be effected by use of an anti-bcl-2 antibody.Such an antibody can be used to immunoprecipitate bcl-2 proteins fromthe set of cellular proteins of the correct approximate molecularweight. Such antibodies can, for example, be raised against polypeptidessynthesized according to the sequence shown in FIGS. 2 and 3.Alternatively, the antibodies can be raised against fusion proteins,which contain bcl-2 sequences as well as those of another protein. Anexample of an antibody raised to a fusion protein is discussed, infra.After immunoprecipitation, the bcl-2 proteins can be released from theantibodies to provide a substantially pure preparation of bcl-2proteins.

If it is desired that bcl-2-alpha (approximately 26 kD) be separatedfrom bcl-2-beta (approximately 22 kD) this separation can be achievedusing either polyacrylamide gels or additional sizing or gel filtrationcolumns. Of course, other separations are possible based on the inherentdifferences between the two proteins at their carboxyl termini.Techniques for using gel fitration columns and immunoprecipitation andantibody releasing are all well known the art.

Any source of B-cells is suitable for use in the diagnostic test of thisinvention. B-cells can be isolated from the lymph nodes. Alternatively,the diagnostic test may employ a sample of peripheral blood obtainedfrom an individual who is being screened for the presence of a B-cellneoplasm. Means for separating B-cells from the peripheral blood arewell known in the art. For example, erythrocytes and granulocytes may beseparated from the B-cells by centrifugation in a liquid having adensity intermediate between the groups of cells to be separated.

Extraction of proteins from B-cells may be performed by any of the manymeans known in the art. For example, cells may be lysed by a detergentor by mechanical means. If desired, nucleic acids can be removed fromthe cell preparation by enzymatic digestion or by precipitation withagents such as streptomycin. Once again, such means are well known inthe art.

Antibodies can be generated which are immunoreactive with the bcl-2proteins by immunization of animals with a fusion protein consisting ofa portion of the beta-galactosidase protein of E. coli and a portion ofthe human bcl-2 proteins. Preferably, the bcl -2 portion will containsequences which are common to both bcl-2-alpha and bcl-2-beta. Ifdesired, such a fusion protein can be purified using the propertieswhich it shares with beta-galactosidase. Anti-sera released against sucha fusion protein in rabbits have been found to be immunoreaetive withboth bcl-2-alpha and bcl-2-beta in vitro. Furthermore, using thisanti-sera immunofluorescent techniques it is possible to determinecellular location of bcl-2 proteins in cells which have been fixed.

Antibodies can also be produced by immunization of animals, such asmice, rabbits and the like, with bcl-2-alpha, bcl-2-beta, fragments ofthem, or both. Alternatively, monoclonal-antibodies can be generatedusing immortalized cell lines to provide uniform and continual antibodysources. Techniques for generating such antibodies are well known in theart. Appropriate antibodies can be screened using the natural geneproducts of bcl-2 or the fusion protein discussed above. While it ispreferred that the antibody used in the diagnostic method immunoreactwith both bcl-2-alpha and beta, an antibody may successfully be usedwhich immunoreacts with only one of them.

The extracted proteins from the B-cells may be contacted with theantibody under suitable conditions for antibody-antigen complexformation. Generally, such conditions are physiological conditions. Theprotein extract may be bound to a solid support such as a nitrocellulosefilter or a microtiter plate.

The antibody will generally bear a "tag" such as a radiolabel, afluorescent label or an enzyme conjugate which under appropriateconditions produces a colored reaction product. Once again, such "tags"are quite well known in the art. Alternatively, if the antibody is nottagged, it can be detected by means of a second antibody from anotherspecies which is reacted with the first antibody. Of course, it ispreferred for means of this diagnostic method that the immunologicaltechnique be as quantitatively sensitive as possible.

Means of detection of the antibody-antigen complexes will depend uponthe method of tagging used for the antibody. For example, radiolabeltags can be detected by autoradiography or scintillation counting, whilethe products of enzyme-linked antibodies can be detectedspectrophotometrically.

A parallel sample to the test sample is employed to provide the control.The control sample consists of an equivalent amount of proteinsextracted from cells, preferably in the same manner as those of the testsample. The amount of protein can readily be determined employingtechniques well known in the art, such as the Lowry or Bradfordtechniques. The cells used for preparing the control sample may beselected from the group consisting of B-cells from a normal human, cellsfrom an established normal B-cell or pre-B-cell line, and non-B-cellsfrom the human who is being screened for the neoplasm.

It is a finding of the present invention that in cases where atranslocation has occurred between chromosomes 14 and 18 ,the level ofbcl-2 protein detected immunologically in the B-cells is at least10-fold higher than the amount of bcl-2 protein found in normal B-cells,in pre-B-cells, or in other non-B-cells from the same human.

To screen for elevated levels of mRNA transcribed from the bcl-2 gene,again one must isolate B-cells from the human who is to be screened. Anyof the many methods known in the art are suitable. Total RNA extractedfrom the B-cells may be used, or alternatively mRNA may be isolated fromthe total cellular RNA. The mRNA may be purified, for example, byaffinity chromotography on oligo(dT) cellulose which binds to thepoly(A) tract at the 3' end of most mRNA. As is well known to those ofskill in the art, it is essential that ribonuclease activity beminimized during preparation and assaying.

A DNA probe may be selected from any of the protein coding sequences ofthe bcl-2 gene. Preferably, the probe will be selected from sequences ofthe 5' or first exon of the gene, so that all three species of RNA canbe detected. Alternatively the probe can be selected from sequenceswhich hybridize exclusively with the 3.5 kb transcript or only with the5.5 kb and the 8.5 kb transcript. Preferably the probe contains at least15 nucleotides of the bcl-2 sequence. Suitable plasmid molecules whichmay be used as probes have been deposited at the ATCC under DepositNumbers 67147 and 67148. Of course, other suitable probes may besynthesized or derived from these or other bcl-2 sequences. In order toperform the hybridization it is desirable that the probe be singlestranded. Thus if the probe is double stranded, it should be denaturedto single stranded form. Means for denaturing are well known in the art,including alkali or heat treatment. The probe can then be contacted withthe RNA derived from the B-cells under conditions where homologousRNA-DNA hybrids form and are stable. Such conditions are well known inthe art. Means for detecting hybrids are many and well known, but ofteninvolve use of radiolabeled probes and nucleases which degrade singlestranded DNA. Other methods may be used.

Control samples can be derived from any of the cell sources describedabove for use in the antibody diagnostic test. Samples and controlshould be prepared in parallel under similar conditions. If comparisonof the test and control sample hybridization shows a greater than abouta ten-fold excess in the test sample, a B-cell neoplasm is indicated.

The following examples do not limit the scope of the invention but aremerely illustrative.

EXAMPLE I

A cDNA library from polyA⁺ mRNA of the pre-B-cell leukemia cell line 380was constructed. Cytoplasmic RNA was extracted by the proceduredescribed in ar-Rushdi, et al (1982) Sematic Cell Genetics, Vol. 8, pp.151-161. PolyA⁺ RNA was Selected by oligo(dT) column chromotography asdescribed in Aviv and Leder, (1972) Proceedings of National Academy ofSciences, USA, Vol. 69, pp. 1408-1412. Double stranded cDNA wassynthesized from mRNA by reverse transcriptase (Life Science, inc.,Florida) using oligo(dT) as primer as described in Maniatis et al (1982)Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. Alter EeoRI linker ligation, the double stranded cDNA was clonedinto lambda gt 11 phage vectors, Young and Davis (1983) Proceedings ofthe National Academy of Sciences, USA, Vol. 80, pp. 1194-1198. Byscreening approximately 2×10⁵ recombinant clones with a DNA probeconsisting of a segment of chromosome 18 which spans the hotspot ofbreak-points of the translocation of chromosome 18 to chromosome 14,three independent cDNA clones were obtained which are overlapping (B3,B4, and B10). As shown in FIG. 1, clone B3 contains nineteen A residuesat the end, indicating that this clone represents the 3' end of themRNA. The restriction maps of the cDNA clones and genomic sequences arecollinear from the 3' end of cDNA clone B3 until lust before the BamHIsite of the cDNA sequence. The cDNA sequence just beyond this pointdiverges from the genomic sequence. Thus, the cDNA sequences consist ofat least two genomic regions.

When the 5' part of cDNA clone B4 (5' end to the BamHI site) is used toprobe the cDNA bank, another set of clones is obtained, including cloneB15 and clone B16 (see FIG. 1). These two cDNA clones have the samesequences at the 5' region as does clone B4, but they have totallydifferent sequences at the 3' region than do clones B3, B4, and B10.Thus cDNA cloning has yielded two different sets of clones, indicatingthat the bcl-2 gene is transcribed into at least two different mRNAs.

In order to obtain cDNA sequences further upstream (in the 5'direction), a cDNA library was constructed using the primer extensionmethod. An otigonucleotide (15-mer) was synthesized and used as a primerfor reverse transcriptase as described in Maniatis, supra, and in Gublerand Hoffman (1983) Gene, Vol. 25, pp. 263-269. Three clones wereobtained by this method, clone B6-3, B22-1, and B9.

EXAMPLE II

Two different probes were used to visualize the mRNA speciescorresponding to the bcl-2 gene in Northern blot hybridizations. Thefirst probe (probe A in FIG. 1) contains genomic DNA of chromosome 18which spans the break-point hotspot and also corresponds to the 3' exon.The other probe used was cDNA clone B22-1 which corresponds to the 5'exon (the first exon). RNA was glyoxalated, run on 1% agarose gels, andblotted to nitrocellulose filters as described in Thomas, Proceedings ofthe National Academy of Sciences, USA (1980) Vol. 77, pp. 5201-5205. Thenitrocellulose filter was hybridized with ³² P-labelled probe in 50%formamide, 4 X SSC, 0.1% SDS at 37° C. and finally washed with 2 X SSC(0.3M NaCl, 0.03 M Na-citrate, pH 7) at 65° C.

The genomic DNA probe A detected two transcripts, 8.5 kb and 5.5 kb inlength. The cDNA probe B22-1 detected the same transcripts as probe A,as well as an additional transcript of 3.5 kb.

The 8.5 kb mRNA was also shown to hybridize to a genomic DNA probe fromchromosome 18 which is 3' to genomic probe A (and is indicated in FIG. 1as probe B).

These data indicate that the bcl-2 gene is transcribed into three mRNAsof different sizes. The possibility that these mRNAs are derived fromdifferent but related genes is excluded by the fact that under the samehybridization conditions which were used for the Northern blothybridization, these probes detect only one cellular gene.

EXAMPLE III

The nucleotide sequence of overlapping cDNA clones was determined byMaxam and Gilbert's chemical degradation method, (Proceedings of theNational Academy or Sciences, USA, Vol. 74, pp. 560-564 (1977), orSanger's chain termination method, Proceedings of the National Academyof Sciences, USA, Vol. 74, pp. 5463-5467 (1977). Both strands of DNAwere sequenced. The nucleotide sequence derived from the 5.5 kbtranscript is shown in FIG. 2. The DNA sequence of 5105 base pairs (bp)reveals one possible open reading frame consisting of 239 amino acidresidues (bcl-2-alpha). The nucleotide sequence corresponding to the 3.5kb transcript is shown in FIG. 3. This transcript codes for a proteinconsisting of 205 amino acid residues (bcl-2-beta), which differs fromthe bcl-2-alpha protein at the carboxyl terminus.

We claim:
 1. A human bcl-2 cDNA molecule.
 2. A polynucleotide comprisinga human bcl-2 cDNA molecule.
 3. The polynucleotide of claim 2 whereinsaid polynucleotide is replicated in bacteria.
 4. The polynucleotide ofclaim 2 wherein said polynucleotide is expressed in bacteria.
 5. A cDNApolynucleotide which comprises the human bcl-2 gene sequence as shown inFIGS. 2 or 3, or the complement thereto.